Search for the best indicators for the presence of a VPS13B gene mutation and confirmation of diagnostic criteria in a series of 34 patients genotyped for suspected Cohen syndrome

BACKGROUND: Cohen syndrome is a rare autosomal recessive inherited disorder that results from mutations of the VPS13B gene. Clinical features consist of a combination of mental retardation, facial dysmorphism, postnatal microcephaly, truncal obesity, slender extremities, joint hyperextensibility, myopia, progressive chorioretinal dystrophy, and intermittent neutropenia.PATIENTS AND METHODS: The aim of the study was to determine which of the above clinical features were the best indicators for the presence of VPS13B gene mutations in a series of 34 patients with suspected Cohen syndrome referred for molecular analysis of VPS13B. RESULTS: 14 VPS13B gene mutations were identified in 12 patients, and no mutation was found in 22 patients. The presence of chorioretinal dystrophy (92% vs 32%, p=0.0023), intermittent neutropenia (92% vs 5%, p<0.001), and postnatal microcephaly (100% vs 48%, p=0.0045) was significantly higher in the group of patients with a VPS13B gene mutation compared to the group of patients without a mutation. All patients with VPS13B mutations had chorioretinal dystrophy and/or intermittent neutropenia. The Kolehmainen diagnostic criteria provided 100% sensibility and 77% specificity when applied to this series. CONCLUSION: From this study and a review of more than 160 genotyped cases from the literature, it is concluded that, given the large size of the gene, VPS13B screening is not indicated in the absence of chorioretinal dystrophy or neutropenia in patients aged over 5 years. The follow-up of young patients could be a satisfactory alternative unless there are some reproductive issues

Global Analysis of Quorum Sensing Targets in the Intracellular Pathogen Brucella melitensis 16 M

Many pathogenic bacteria use a regulatory process termed quorum sensing (QS) to produce and detect small diffusible molecules to synchronize gene expression within a population. In Gram-negative bacteria, the detection of, and response to, these molecules depends on transcriptional regulators belonging to the LuxR family. Such a system has been discovered in the intracellular pathogen Brucella melitensis, a Gram-negative bacterium responsible for brucellosis, a worldwide zoonosis that remains a serious public health concern in countries were the disease is endemic. Genes encoding two LuxR-type regulators, VjbR and BabR, have been identified in the genome of B. melitensis 16 M. A DeltavjbR mutant is highly attenuated in all experimental models of infection tested, suggesting a crucial role for QS in the virulence of Brucella. At present, no function has been attributed to BabR. The experiments described in this report indicate that 5% of the genes in the B. melitensis 16 M genome are regulated by VjbR and/or BabR, suggesting that QS is a global regulatory system in this bacterium. The overlap between BabR and VjbR targets suggest a cross-talk between these two regulators. Our results also demonstrate that VjbR and BabR regulate many genes and/or proteins involved in stress response, metabolism, and virulence, including those potentially involved in the adaptation of Brucella to the oxidative, pH, and nutritional stresses encountered within the host. These findings highlight the involvement of QS as a major regulatory system in Brucella and lead us to suggest that this regulatory system could participate in the spatial and sequential adaptation of Brucella strains to the host environment.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Expression of VjbR under Nutrient Limitation Conditions Is Regulated at the Post-Transcriptional Level by Specific Acidic pH Values and Urocanic Acid

VjbR is a LuxR homolog that regulates transcription of many genes including important virulence determinants of the facultative intracellular pathogen Brucella abortus. This transcription factor belongs to a family of regulators that participate in a cell-cell communication process called quorum sensing, which enables bacteria to respond to changes in cell population density by monitoring concentration of self produced autoinducer molecules. Unlike almost all other LuxR-type proteins, VjbR binds to DNA and activates transcription in the absence of any autoinducer signal. To investigate the mechanisms by which Brucella induces VjbR-mediated transcriptional activation, and to determine how inappropriate spatio-temporal expression of the VjbR target genes is prevented, we focused on the study of expression of vjbR itself. By assaying different parameters related to the intracellular lifestyle of Brucella, we identified a restricted set of conditions that triggers VjbR protein expression. Such conditions required the convergence of two signals of different nature: a specific pH value of 5.5 and the presence of urocanic acid, a metabolite involved in the connection between virulence and metabolism of Brucella. In addition, we also observed an urocanic acid, pH-dependent expression of RibH2 and VirB7, two additional intracellular survival-related proteins of Brucella. Analysis of promoter activities and determination of mRNA levels demonstrated that the urocanic acid-dependent mechanisms that induced expression of VjbR, RibH2, and VirB7 act at the post-transcriptional level. Taken together, our findings support a model whereby Brucella induces VjbR-mediated transcription by modulating expression of VjbR in response to specific signals related to the changing environment encountered within the host

Morphological analysis of the sheathed flagellum of Brucella melitensis

<p>Abstract</p> <p>Background</p> <p>It was recently shown that <it>B. melitensis </it>is flagellated. However, the flagellar structure remains poorly described.</p> <p>Findings</p> <p>We analyzed the structure of the polar sheathed flagellum of <it>B. melitensis </it>by TEM analysis and demonstrated that the Ryu staining is a good method to quickly visualize the flagellum by optical microscopy. The TEM analysis demonstrated that an extension of the outer membrane surrounds a filament ending by a club-like structure. The Δ<it>ftcR</it>, Δ<it>fliF</it>, Δ<it>flgE </it>and Δ<it>fliC </it>flagellar mutants still produce an empty sheath.</p> <p>Conclusions</p> <p>Our results demonstrate that the flagellum of <it>B. melitensis </it>has the characteristics of the sheathed flagella. Our results also suggest that the flagellar sheath production is not directly linked to the flagellar structure assembly and is not regulated by the FtcR master regulator.</p

Genome Degradation in Brucella ovis Corresponds with Narrowing of Its Host Range and Tissue Tropism

Brucella ovis is a veterinary pathogen associated with epididymitis in sheep. Despite its genetic similarity to the zoonotic pathogens B. abortus, B. melitensis and B. suis, B. ovis does not cause zoonotic disease. Genomic analysis of the type strain ATCC25840 revealed a high percentage of pseudogenes and increased numbers of transposable elements compared to the zoonotic Brucella species, suggesting that genome degradation has occurred concomitant with narrowing of the host range of B. ovis. The absence of genomic island 2, encoding functions required for lipopolysaccharide biosynthesis, as well as inactivation of genes encoding urease, nutrient uptake and utilization, and outer membrane proteins may be factors contributing to the avirulence of B. ovis for humans. A 26.5 kb region of B. ovis ATCC25840 Chromosome II was absent from all the sequenced human pathogenic Brucella genomes, but was present in all of 17 B. ovis isolates tested and in three B. ceti isolates, suggesting that this DNA region may be of use for differentiating B. ovis from other Brucella spp. This is the first genomic analysis of a non-zoonotic Brucella species. The results suggest that inactivation of genes involved in nutrient acquisition and utilization, cell envelope structure and urease may have played a role in narrowing of the tissue tropism and host range of B. ovis

PLS3 Missense Variants Affecting the Actin-Binding Domains Cause X-Linked Congenital Diaphragmatic Hernia and Body-Wall Defects

Congenital diaphragmatic hernia (CDH) is a relatively common and genetically heterogeneous structural birth defect associated with high mortality and morbidity. We describe eight unrelated families with an X-linked condition characterized by diaphragm defects, variable anterior body-wall anomalies, and/or facial dysmorphism. Using linkage analysis and exome or genome sequencing, we found that missense variants in plastin 3 (PLS3), a gene encoding an actin bundling protein, co-segregate with disease in all families. Loss-of-function variants in PLS3 have been previously associated with X-linked osteoporosis (MIM: 300910), so we used in silico protein modeling and a mouse model to address these seemingly disparate clinical phenotypes. The missense variants in individuals with CDH are located within the actin-binding domains of the protein but are not predicted to affect protein structure, whereas the variants in individuals with osteoporosis are predicted to result in loss of function. A mouse knockin model of a variant identified in one of the CDH-affected families, c.1497G\u3eC (p.Trp499Cys), shows partial perinatal lethality and recapitulates the key findings of the human phenotype, including diaphragm and abdominal-wall defects. Both the mouse model and one adult human male with a CDH-associated PLS3 variant were observed to have increased rather than decreased bone mineral density. Together, these clinical and functional data in humans and mice reveal that specific missense variants affecting the actin-binding domains of PLS3 might have a gain-of-function effect and cause a Mendelian congenital disorder

Brucellosis Vaccines: Assessment of Brucella melitensis Lipopolysaccharide Rough Mutants Defective in Core and O-Polysaccharide Synthesis and Export

Background: The brucellae are facultative intracellular bacteria that cause brucellosis, one of the major neglected zoonoses. In endemic areas, vaccination is the only effective way to control this disease. Brucella melitensis Rev 1 is a vaccine effective against the brucellosis of sheep and goat caused by B. melitensis, the commonest source of human infection. However, Rev 1 carries a smooth lipopolysaccharide with an O-polysaccharide that elicits antibodies interfering in serodiagnosis, a major problem in eradication campaigns. Because of this, rough Brucella mutants lacking the O-polysaccharide have been proposed as vaccines. Methodology/Principal Findings: To examine the possibilities of rough vaccines, we screened B. melitensis for lipopolysaccharide genes and obtained mutants representing all main rough phenotypes with regard to core oligosaccharide and O-polysaccharide synthesis and export. Using the mouse model, mutants were classified into four attenuation patterns according to their multiplication and persistence in spleens at different doses. In macrophages, mutants belonging to three of these attenuation patterns reached the Brucella characteristic intracellular niche and multiplied intracellularly, suggesting that they could be suitable vaccine candidates. Virulence patterns, intracellular behavior and lipopolysaccharide defects roughly correlated with the degree of protection afforded by the mutants upon intraperitoneal vaccination of mice. However, when vaccination was applied by the subcutaneous route, only two mutants matched the protection obtained with Rev 1 albeit at doses one thousand fold higher than this reference vaccine. These mutants, which were blocked in O-polysaccharide export and accumulated internal O-polysaccharides, stimulated weak anti-smooth lipopolysaccharide antibodies. Conclusions/Significance: The results demonstrate that no rough mutant is equal to Rev 1 in laboratory models and question the notion that rough vaccines are suitable for the control of brucellosis in endemic areas.This work was funded by the European Commission (Research Contract QLK2-CT-2002-00918) and the Ministerio de Ciencia y Tecnología of Spain (Proyecto AGL2004-01162/GAN)